A preservation solution for extending the low-temperature storage time of NK cells, and its preparation method and application

Abstract

The present invention relates to a preservation solution for extending the low-temperature preservation time of NK cells, a preparation method thereof, and an application thereof. The components of the preservation solution include: dextran 40 glucose injection, 10% glucose injection, 50% glucose injection, 20% human serum albumin, compound amino acid injection, trehalose, coenzyme Q10, mulberry flavonoids, and normal saline. The present invention uses compound amino acid injection supplemented with three specific active substances, trehalose, coenzyme Q10, and mulberry flavonoids, wherein trehalose can achieve significantly excellent NK cell low-temperature preservation effects compared with other sugar substances; wherein coenzyme Q10 and mulberry flavonoids play a role through antioxidant and anti-apoptotic properties, can maintain the metabolic activity of NK cells and reduce oxidative damage, and the two have a significant synergistic promoting effect in improving the low-temperature preservation effect of NK cells. The components in the preservation solution of the present invention are mutually formulated to achieve an excellent NK cell low-temperature preservation effect as a whole.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell preservation, and relates to a preservation solution for prolonging the low-temperature preservation time of NK cells, and a preparation method and application thereof. Background Art

[0002] As the core effector cells of the innate immune system, NK cells can recognize and eliminate tumor cells without antigen presensitization, especially against "immune escape" tumor cells with downregulated MHC-I. They have shown great potential in tumor immunotherapy, but their clinical application faces many bottlenecks.

[0003] Traditional freezing and thawing methods are prone to cell damage. Traditional liquid nitrogen freezing uses DMSO (dimethyl sulfoxide) as a cryoprotectant, resulting in a significant decrease in cell viability and function after thawing. CN118489672A discloses a NK cell freezing solution that uses N-tris(hydroxymethyl)methylglycine and sodium glycinate as permeable cryoprotectants, and carboxymethyl chitosan, polyglutamic acid, and sodium polyglutamate as non-permeable cryoprotectants. These two components are blended with culture medium to form a freezing solution for NK cell cryopreservation. This solution, without the addition of DMSO or fetal bovine serum, achieves NK cell cryopreservation through the synergistic effect of these two agents. However, this freezing solution is only suitable for liquid nitrogen freezing and cannot be used for low-temperature storage (2-8°C). Therefore, it cannot be used for low-temperature transportation, significantly limiting its scope of application.

[0004] CN118661721A discloses a cryopreservation solution for cryopreserving NK cells at low temperatures. By adding hydroxyethyl starch, cedarwood glycol, and butyl ferulate to the culture medium, this solution enables long-term storage of NK cells in liquid nitrogen. NK cells stored in this cryopreservation solution for 15 months still maintain high cell viability and cytotoxic activity. However, 24 hours after NK cell recovery, the viability drops sharply to 60%-70%, and the early apoptosis rate (Annexin V⁺) is as high as 25%-35%. This means that NK cells must be used immediately after recovery, which does not address the long-distance transportation problem of NK cells.

[0005] CN118370310A discloses a NK cell cryopreservation solution containing dextran, a ROCK inhibitor, and IL-7. The combination of IL-7, dextran, and the ROCK inhibitor can prevent NK cell apoptosis caused by cryopreservation and maintain high cytotoxic activity after cryopreservation. However, this cryopreservation solution is only suitable for liquid nitrogen freezing and cannot be stored at low temperatures (2-8°C). Therefore, it cannot be used for low-temperature transportation, which greatly limits its application.

[0006] The low-temperature (2-8°C) NK cell preservation solution disclosed in the existing technology (such as the electrolyte solution containing Promali A) can only maintain the viability of NK cells for about 24 hours. After 24 hours, the viability drops to below 60%. To ensure cell activity, clinicians are forced to use air transportation and real-time temperature control, which increases transportation and treatment costs, and cannot cover remote areas, and the scope of application is very limited.

[0007] Therefore, it would be very meaningful to develop a method that can preserve NK cells at low temperatures (2-8°C) and maintain a high viability and tumor-killing activity of the cells after long-term storage. Summary of the Invention

[0008] To address the shortcomings of the existing technology, the present invention aims to provide a preservation solution for extending the cryopreservation time of NK cells, as well as its preparation method and application. This preservation solution can extend the storage time of NK cells to more than 10 days at 2-8°C while maintaining a high viability and tumor-killing activity of NK cells.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a preservation solution for prolonging the low-temperature storage time of NK cells, wherein the components of the preservation solution include: dextran 40 glucose injection, 10% glucose injection, 50% glucose injection, 20% human serum albumin, compound amino acid injection, trehalose, coenzyme Q10, mulberry flavonoids and normal saline.

[0011] The components in the NK cell preservation solution of the present invention can all be derived from commercially available raw materials. These raw materials all meet injection-grade safety requirements and can be used directly by injection.

[0012] The present invention replaces the electrolyte solutions such as Bomeili used in the prior art with a compound amino acid injection to provide cellular energy metabolism support and anti-oxidative damage, thereby avoiding the acetate toxicity caused by Bomeili.

[0013] At the same time, the preservation solution is supplemented with three specific active substances: trehalose, coenzyme Q10, and mulberry flavone. Among them, trehalose can reduce the phase change of membrane lipids at low temperatures, avoiding damage caused by sudden changes in membrane fluidity; it can stabilize the native conformation of proteins, prevent denaturation or aggregation, maintain enzyme activity and signal pathway function; it can inhibit oxidative stress response and reduce damage to cells by reactive oxygen species (ROS); and for the preservation solution formula of the present invention, trehalose can achieve significantly better NK cell low-temperature preservation effect compared with other sugar substances (such as sucrose, mannitol, lactose, maltose, fructose, etc.).

[0014] Among them, coenzyme Q10 and mulberry flavone mainly exert their effects through their antioxidant and anti-apoptotic properties, which can maintain the metabolic activity of NK cells and reduce oxidative damage. The two have obvious synergistic promoting effects in improving the low-temperature preservation effect of NK cells.

[0015] Preferably, the volume fraction of the dextran 40 glucose injection in the preservation solution is 15-20%, for example, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, etc. Other specific point values ​​within this numerical range can be selected and will not be repeated here.

[0016] Preferably, the volume fraction of the 10% glucose injection in the preservation solution is 2-5%, for example, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0017] Preferably, the volume fraction of the 50% glucose injection in the preservation solution is 0.5-1%, for example, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0018] Preferably, the volume fraction of the 20% human serum albumin in the preservation solution is 22-28%, for example, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 28%, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0019] Preferably, the volume fraction of the compound amino acid injection in the preservation solution is 28-34%, for example, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 34%, etc. Other specific point values ​​within this numerical range can be selected and will not be repeated here.

[0020] Preferably, the final concentration of trehalose in the preservation solution is 10-15 mM, for example, 10 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, 15 mM, etc. Other specific values ​​within this numerical range can be selected and will not be described here one by one.

[0021] Based on the irreplaceable nature of trehalose in the preservation solution formulation of the present invention, its final concentration in the preservation solution also affects the preservation effect of the preservation solution to a certain extent, with 10-15 mM being a more preferred range.

[0022] Preferably, the final concentration of the coenzyme Q10 in the preservation solution is 15-25 μM, for example, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, etc. Other specific points within this numerical range can be selected and will not be described here one by one.

[0023] Preferably, the final concentration of mulberry flavone in the preservation solution is 5-10 μM, for example, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, etc. Other specific points within this numerical range can be selected and will not be described here one by one.

[0024] Based on the synergistic effect of coenzyme Q10 and mulberry flavone in improving the low-temperature preservation effect of NK cells, the effect is better when they meet the above preferred final concentration range.

[0025] Preferably, the compound amino acid injection is selected from any one of compound amino acid injection (9AA), compound amino acid injection (14AA), compound amino acid injection (15AA), compound amino acid injection (17AA), compound amino acid injection (18AA) or compound amino acid injection (20AA).

[0026] The above-mentioned various types of compound amino acid injections are all commercially available clinical injections.

[0027] Preferably, the osmotic pressure of the preservation solution is 280-350 mOsm / kg, for example, 280 mOsm / kg, 290 mOsm / kg, 295 mOsm / kg, 300 mOsm / kg, 310 mOsm / kg, 320 mOsm / kg, 330 mOsm / kg, 340 mOsm / kg, 350 mOsm / kg, etc.; the pH is 7.0-7.5, for example, pH=7.0, pH=7.1, pH=7.2, pH=7.3, pH=7.4, pH=7.5, etc.; other specific point values ​​within the above numerical range can be selected and will not be repeated here.

[0028] In a second aspect, the present invention provides a method for preparing the preservation solution for prolonging the low-temperature storage time of NK cells according to the first aspect, the preparation method comprising the following steps:

[0029] Mix dextran 40% glucose injection, 10% glucose injection, 50% glucose injection, 20% human serum albumin, compound amino acid injection, and normal saline; then add trehalose, coenzyme Q10, and mulberry flavonoids to the mixed solution, and filter through a 0.22 μm filter membrane after dissolution to obtain the product.

[0030] Preferably, the mulberry flavone is added to the preservation solution in the form of DMSO stock solution, and the final concentration of DMSO is ≤0.1%.

[0031] In a third aspect, the present invention provides a use of the preservation solution according to the first aspect in preserving NK cells at 2-8° C. After the NK cells are stored at 2-8° C. for 10 days, the cell viability and cytotoxicity against K562 cells remain at a high level.

[0032] Preferably, the NK cells include any one of peripheral blood-derived NK cells, umbilical cord blood-derived NK cells, iPSC-derived NK cells or CAR-NK cells.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention replaces the electrolyte solutions such as Bomeili used in the prior art with a compound amino acid injection to provide cellular energy metabolism support and anti-oxidative damage, thereby avoiding the acetate toxicity caused by Bomeili.

[0035] At the same time, the preservation solution is supplemented with three specific active substances: trehalose, coenzyme Q10, and mulberry flavonoids. Trehalose is irreplaceable for the preservation solution formula of the present invention. Compared with other sugar substances (such as sucrose, mannitol, lactose, maltose, fructose, etc.), trehalose can achieve significantly better NK cell cryopreservation effect; among them, coenzyme Q10 and mulberry flavonoids mainly play a role through antioxidant and anti-apoptotic properties, which can maintain the metabolic activity of NK cells and reduce oxidative damage. The two have a significant synergistic effect in improving the low-temperature preservation effect of NK cells. The various components in the preservation solution of the present invention are formulated together to achieve an excellent NK cell cryopreservation effect as a whole. DETAILED DESCRIPTION

[0036] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0037] The sources of some of the following preparation raw materials are shown in Table 1:

[0038] Table 1

[0039]

[0040] Example 1

[0041] This embodiment provides a NK cell preservation solution, the components of which include the following:

[0042] Dextran 40 glucose injection (16.67% by volume), 10% glucose injection (3.12% by volume), 50% glucose injection (0.83% by volume), 20% human serum albumin (25% by volume), compound amino acid injection (15AA) (31.25% by volume), trehalose (final concentration 12 mM), coenzyme Q10 (final concentration 20 μM), DMSO solution of mulberry flavonoids (final concentration of mulberry flavonoids 8 μM, DMSO volume ratio is 0.05%), and the balance is normal saline.

[0043] The preparation method is as follows: the components are mixed in proportion to fully dissolve the active component, the mixture is sterilized by filtering through a 0.22 μm filter membrane, and the mixture is stored at 4°C away from light.

[0044] Example 2

[0045] This embodiment provides a NK cell preservation solution, the components of which include the following:

[0046] Dextran 40 glucose injection (16.7% by volume), 10% glucose injection (3.15% by volume), 50% glucose injection (0.86% by volume), 20% human serum albumin (25.5% by volume), compound amino acid injection (17AA) (31.75% by volume), trehalose (final concentration 15 mM), coenzyme Q10 (final concentration 23 μM), DMSO solution of mulberry flavonoids (final concentration of mulberry flavonoids 5 μM, volume proportion of DMSO is 0.05%), and the balance is normal saline.

[0047] The preparation method is as follows: the components are mixed in proportion to fully dissolve the active component, the mixture is sterilized by filtering through a 0.22 μm filter membrane, and the mixture is stored at 4°C away from light.

[0048] Example 3

[0049] This embodiment provides a NK cell preservation solution, the components of which include the following:

[0050] Dextran 40 glucose injection (17.07% by volume), 10% glucose injection (3.16% by volume), 50% glucose injection (0.87% by volume), 20% human serum albumin (25.8% by volume), compound amino acid injection (18AA) (31.75% by volume), trehalose (final concentration 10 mM), coenzyme Q10 (final concentration 15 μM), DMSO solution of mulberry flavonoids (final concentration of mulberry flavonoids 10 μM, volume proportion of DMSO is 0.05%), and the balance is normal saline.

[0051] The preparation method is as follows: the components are mixed in proportion to fully dissolve the active component, the mixture is sterilized by filtering through a 0.22 μm filter membrane, and the mixture is stored at 4°C away from light.

[0052] Example 4

[0053] This example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in that the final concentration of mulberry flavone is 3 μM, while the other components remain unchanged. The preparation method thereof is similar to that of Example 1.

[0054] Example 5

[0055] This example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in that the final concentration of mulberry flavone is 12 μM, while the other components remain unchanged. The preparation method thereof is similar to that of Example 1.

[0056] Comparative Example 1

[0057] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in the absence of coenzyme Q10, with the other components remaining unchanged. The preparation method thereof is similar to that of Example 1.

[0058] Comparative Example 2

[0059] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in the absence of the addition of mulberry flavone, while the other components remain unchanged. The preparation method thereof is similar to that of Example 1.

[0060] Comparative Example 3

[0061] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in the absence of coenzyme Q10 and a final concentration of 10 μM mulberry flavonoids, with the other components remaining unchanged. The preparation method thereof is similar to that of Example 1.

[0062] Comparative Example 4

[0063] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in the absence of mulberry flavone and a final concentration of coenzyme Q10 of 25 μM, with the other components remaining unchanged. The preparation method thereof is similar to that of Example 1.

[0064] Comparative Example 5

[0065] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in that trehalose is replaced with sucrose at equal concentrations, while the other components remain unchanged. The preparation method thereof refers to that of Example 1.

[0066] Comparative Example 6

[0067] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in that trehalose is replaced with mannitol at equal concentrations, while the other components remain unchanged. The preparation method thereof refers to that of Example 1.

[0068] Comparative Example 7

[0069] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in that trehalose is replaced with maltose at equal concentrations, while the other components remain unchanged. The preparation method thereof refers to that of Example 1.

[0070] Comparative Example 8

[0071] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in that trehalose is replaced with lactose at equal concentrations, while the other components remain unchanged. The preparation method thereof refers to that of Example 1.

[0072] Comparative Example 9

[0073] This comparative example provides a NK cell preservation solution, the components of which differ from those of Example 1 only in the absence of trehalose, with the other components remaining unchanged. The preparation method thereof is similar to that of Example 1.

[0074] Comparative Example 10

[0075] This comparative example provides a NK cell preservation solution. The components of this solution differ from those of Example 1 only in that an equal volume of compound amino acid injection (15AA) is replaced with compound electrolyte injection (Bomeili A). All other components remain unchanged. The preparation method is similar to that of Example 1.

[0076] Test Case

[0077] The cell preservation solution prepared in Examples 1-5 and Comparative Examples 1-10 was used to preserve NK cells (CD56+CD3- purity > 95%) from the peripheral blood of healthy volunteers on the 14th day of expansion culture at 4°C. The specific operation was as follows: NK cells were harvested by centrifugation, the supernatant was discarded, the cells were resuspended in physiological saline and centrifuged again for washing, the supernatant was discarded, and the prepared cell preservation solution was used to store 5×10 7 Each bag was filled with 100 mL of NK cells. A commercial NK preservation solution (StemSpan™ SF) was used as a control.

[0078] (1) The cell activity of each group was detected on the 1st, 3rd, 5th, 7th, and 10th day of storage using a Counterstar cell counter and AOPI staining. The results are shown in Table 2.

[0079] (2) The cytotoxicity of NK cells from each group on days 1, 3, 5, 7, and 10 against K562 cells (human chronic myeloid leukemia cells) (at an effector-target ratio of 5:1) was tested. The test method was as follows: NK cells and K562 cells were co-incubated at a 37°C CO2 incubator for 4 h at an effector-target ratio of 5:1, and the cytotoxicity was tested by the MTT assay. The results are shown in Table 3.

[0080] Table 2

[0081]

[0082] Table 3

[0083]

[0084] As can be seen from the data results in Tables 2 and 3, the preservation solution involved in the present invention can achieve excellent NK cell low-temperature preservation effect. The preservation solution can extend the storage time of NK cells to 10 days under 2-8°C conditions, while maintaining a high viability and tumor killing activity of NK cells.

[0085] By comparing Example 1 with Comparative Example 10, it can be seen that the present invention replaces the electrolyte solution such as Bomeili used in the prior art with the compound amino acid injection to provide cellular energy metabolism support and anti-oxidative damage, thereby avoiding the acetate toxicity caused by Bomeili.

[0086] By comparing Example 1 with Comparative Examples 5-9, the importance of trehalose in the preservation solution of the present invention can be seen. In addition, for the preservation solution formula of the present invention, trehalose can achieve significantly better NK cell cryopreservation effect compared with other commonly used sugars.

[0087] By comparing Example 1 with Comparative Examples 1-4, it can be seen that the importance of coenzyme Q10 and mulberry flavone in the preservation solution of the present invention, and the two have an obvious synergistic promoting effect in improving the low-temperature preservation effect of NK cells.

[0088] By comparing Example 1 with Examples 4-5, it can be seen that the final concentration of mulberry flavone in the preservation solution also affects the cryopreservation effect of NK cells to a certain extent. When the final concentration of mulberry flavone in the preservation solution is 5-10 μM, the NK cells have a better cryopreservation effect.

[0089] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0090] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0091] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A preservation solution for prolonging the low-temperature storage time of NK cells at 2-8°C, characterized in that: The components of the preservation solution are: 15-20% dextran 40 glucose injection, 2-5% 10% glucose injection, 0.5-1% 50% glucose injection, 22-28% 20% human serum albumin, 28-34% compound amino acid injection, 10-15 mM trehalose, 15-25 μM coenzyme Q10, 5-10 μM mulberry flavonoids and normal saline.

2. The preservation solution for prolonging the low-temperature storage time of NK cells according to claim 1, characterized in that The compound amino acid injection is selected from any one of compound amino acid injection 9AA, 14AA, 15AA, 17AA, 18AA or 20AA.

3. The preservation solution for prolonging the low-temperature storage time of NK cells according to claim 1, characterized in that The osmotic pressure of the preservation solution is 280-350 mOsm / kg, and the pH is 7.0-7.

5.

4. The method for preparing a preservation solution for prolonging the low-temperature storage time of NK cells according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: Mix dextran 40% glucose injection, 10% glucose injection, 50% glucose injection, 20% human serum albumin, compound amino acid injection, and normal saline; then add trehalose, coenzyme Q10, and mulberry flavonoids to the mixed solution, and filter through a 0.22 μm filter membrane after dissolution to obtain the product.

5. Use of the preservation solution according to any one of claims 1 to 3 for preserving NK cells at 2-8°C.

6. The use according to claim 5, characterized in that The NK cells include any one of peripheral blood-derived NK cells, umbilical cord blood-derived NK cells, iPSC-derived NK cells or CAR-NK cells.

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